Annexin A3 for cancer diagnosis

ABSTRACT

A method treats urogenital and/or intestinal tract cancer and includes administering a therapeutically effective amount of at least one annexion protein, annexin of A3, to a mammal.

RELATED APPLICATIONS

This application is a divisional of U.S. Application Ser. No. 11/920,822filed Nov. 20, 2007, issued as U.S. Pat. No. 7,732,148, which is a §371of International Application No. PCT/EP2006/004818, with aninternational filing date of May 22, 2006 (WO 2006/125580 A1, publishedNov. 30, 2006), which is based on European Patent Application Nos.05011042.8, filed May 21, 2005, and 05026092.6, filed Nov. 30, 2005.

TECHNICAL FIELD

This disclosure relates to the treatment and/or diagnosis of cancer,particularly of the urogenital and/or intestinal tract.

BACKGROUND

Cancer is one of the leading causes of human death in the westerncivilization and often linked with difficulties regarding its diagnosis.

For example, prostate cancer is one of the leading causes of cancerdeath in men but is a heterogeneous disease that is difficult todiagnose. Predicting the course that an individual tumor will take isalmost impossible. The current state of diagnostic prostate cancermarkers is essentially based on different isoforms of prostate specificantigen (PSA) and on the whole is not sactisfactory in terms of falsenegatives and false positives. (1-4). Recently, various alternativemolecular markers have been suggested from body fluids or prostatetissue (5-11). At least three different subclasses of prostate cancerhave been identified that seem related to tumor grade, incidence ofrecurrence, and metastases (12). Fatty acid synthase alone definesdistinct molecular signatures for prostate cancer (13). Yet, there isurgent remaining need for more elaborate and reliable therapeutic anddiagnostic parameters to characterize patients according to their riskof progression to develop novel appropriate multimodal therapystrategies for improved individual cancer control (14-16).

SUMMARY

We provide a method of treating urogenital and/or intestinal tractcancer including administering a therapeutically effective amount of atleast one annexin protein, annexin of A3, to a mammal.

We also provide a method of diagnosing urogenital and/or intestinaltract cancer and/or discrimination between cancerous and non-canceroustissue, including determining abundance of at least one intracellularannexin protein and/or determining abundance of at least oneextracellular annexin protein, with urine samples or fractions thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a characterization of the anti-annexin A3 rabbit polyclonalantiserum by Western blot. (A) Western blot of 1-D SDS gel. Lane 1:Magic Mark (Invitrogen) molecular weight markers 3 μl per lane, givesmasses in kDa of 120, 100, 80, 60, 50, 40, 30, and 20. Replicate lanescontain 15 μg whole tissue protein extract from cancer (Lanes 2-4) andbenign (Lanes 5-7) prostate from patient 29. The filter was incubatedusing anti annexin 3 polyclonal serum (1:20,000). Lanes 1-7 show a falsecolor image of signal from the entire filter. The boxed insert (Lanes 1′and 2′) shows the single color depiction of lanes 1 and 2. The positionsof annexin A3 and resumed annexin A6 bands are indicated. (B) 2D-PAGEWestern blot of protein 100 μg extract from the cancer sample of patient29 in false color shows the distribution of proteins with crossreactivity to the polyclonal serum. The identity of three strongest (redcolored) annexin A3 spots was confirmed by MALDI-TOF PMF (data notshown), the strongest of which corresponds to the protein spot detectedin our proteomics analysis.

FIG. 2: Immune histochemistry of Annexin 3:annexin A3 immunoreactivityof the anti-annexin A3 polyclonal serum (dilution: 1:100) was found inthe epithelial cells of A) benign prostate tissue. In B) cribbriformprostatic intraepithelial neoplasis (PIN) and C) cancer tissue,epithelial and cancer cells were stained. An elevated level of diffuseextra-epithelial localisation was also observed. Brown color indicatesannexin A3-specific peroxidase staining. Blue color represents counterstaining of the tissue with Gill's hematoxylin solution (Sigma).

FIG. 3: Western blot quantification of annexin A3 in exprimate pelletsof urine obtained after prostatic massage of cancer patients. The toppanels show the chemiluminescent annexin A3 signal from blottedproteins. The bottom panels show the loaded protein stained with PonceauS (‘Protein’). Each gel contains a molecular weight ladder (M) as wellas duplicates of 7.5 μg of total cell protein lysate from a prostatetumor containing annexin A3 as a positive control (+C).

FIG. 4: Western blot, quantification of annexin A3 in exprimate pelletsof urine obtained after prostatic massage of BPH patients. Other detailsfollow FIG. 3.

FIG. 5: Western blot quantification of annexin A3 in exprimate pellet ofurine obtained after prostatic massage of non-cancer control patients.Other details follow FIG. 3.

FIG. 6: Normalized annexin A3 signals from cell pellets of urine ofpatients following prostate massage as shown in FIG. 3 to FIG. 5.Annexin A3 values from different gels are normalized in arbitrary unitsof 7.5 μg of total cell protein lysate from a prostate tumor (PR-26CA)containing annexin A3 as a positive control in each of FIG. 3 to FIG. 5.

FIG. 7: Ratio of annexin A3 content between EDTA-treated supernatantsand cell pellets of exprimate urine samples of patients followingprostatic massage (additional patient material, not contained in FIG.6); a.u. are arbitrary units.

FIG. 8: ROC Curve—Two-Step Procedure 1:

-   -   “Use U_ANX_tot if 2.5≦PSA_ini≦12, most obvious decision        otherwise”

Legend:

-   -   VARIABLE=IF(PSA_ini<2.5; 100000; IF(PSA_ini<=12; u_anx_tot; 0))        twostepvar1    -   CLASSIFICATION VARIABLE: PCa    -   POSITIVE GROUP: PCa=1    -   Sample size=137    -   NEGATIVE GROUP: PCa=0    -   Sample size=101    -   Disease prevalence (%)=57.6    -   Area under the ROC curve=0.740    -   Standard error=0.033    -   95% Confidence interval=0.679 to 0.794    -   P (Area=0.5)<0.0001

FIG. 9: ROC Curve—T comb.var.anx.psa2:≡−2.386+3.294 log(1+PSA_ini)−0.475 log(1+PU_ANX tot)

Legend:

-   -   select: AND(PSA_ini>=2; PSA_ini<=6)***    -   classification variable PCa    -   positive group: PCa=1, Sample size=57    -   negative group: PCa=0, Sample size=52    -   Disease prevalence (%)=52.3    -   Area under the ROC curve=0.791    -   Standard error=0.043    -   95% Confidence interval=0.703 to 0.863    -   P (Area=0.5)<0.0001

DETAILED DESCRIPTION

In view thereof, we provide in a first aspect by the use of at least oneannexin protein, preferably annexin of A3, for the treatment of cancer,particularly of the urogenital and/or intestinal tract, preferably ofprostate cancer.

We also provide for the use of at least one annexin protein, preferablyof annexin A3, for the manufacture of a medicament for treatment ofcancer, particularly of the urogenital and/or intestinal tract,preferably of prostate cancer. In a preferred aspect, cancer treatmentis done by the enhancement of the in vivo abundance of at least oneannexin protein, in particular, by the enhancement of the in vivoabundance of at least one extracellular annexin protein.

We further provide methods for diagnosing cancer, particularly of theurogenital and/or intestinal tract, and/or for discrimination betweencancerous and non-cancerous tissue comprising the separate steps of:

-   -   determining the intracellular abundance of at least one annexin        protein and/or    -   determining the extracellular abundance of at least one annexin        protein, in particular using urine samples or fractions thereof.

The method also comprises the separate steps of:

-   -   determining the intracellular abundance of at least one annexin        protein and    -   determining the extracellular abundance of at least one annexin        protein, in particular using urine samples or fractions thereof.

The method may also comprise the separate steps of

-   -   determining the intracellular abundance of at least one annexin        protein and    -   determining the extracellular abundance of at least one said        annexin protein,        in particular using urine samples or fractions thereof.

After determination of the intra- and extracellular abundance of atleast one annexin protein ratios of the extracellular abundance over theintracellular abundance or the other way around may be determined.Preferably, ratios of the extracellular abundance over the intracellularabundance are determined. The obtained ratios are advantageousdiagnostic parameters for cancer and/or for discrimination betweencancerous and non-cancerous tissue.

The term “extracellular” is understood as the extracellular spaceincluding the outer surface of plasma membranes of cells.

The term “non-cancerous tissue” comprises healthy tissue and pathogenictissue, in particular benign prostatic hyperplasia, chronic prostatitis,Crohn's disease, colitis ulcerosa, inflammable tissue and fibroses, inparticular secondary fibroses.

The term “abundance” is understood as the intracellular and/orextracellular level and concentration respectively of a protein.

The term “annexin protein” and “protein” in general comprise isoforms,mutants, truncated versions and post-translational modified formsthereof. Post-translational modified forms can in particular includeproteinaceous forms obtainable by proteolytic processing.

The term “treatment” is equivalent to “therapy,” thus comprising thetreatment of troubles associated with cancer.

The annexin protein may at least be a member of the group consisting ofannexin A1, annexin A2, annexin A3, annexin A4, annexin A5, annexin A6,annexin A7, annexin A8 and annexin A10 and wherein preferably theabundance of the at least one annexin protein is determined togetherwith the abundance of at least a further protein. With respect to thefurther protein it is referred to the following description.

The abundance of at least one annexin protein may be determined togetherwith the abundance of a small molecule or nucleic acid marker.

Annexins are calcium-binding proteins thought to influence variousintra- and extra-cellular functions, including membrane trafficking,lymphocyte migration, cell motility, calcium flux, and signaltransduction. They are highly abundant, and the calcium-dependent bulkmasking of negatively charged membrane lipids may be important forannexin function (17).

In a previous proteomics study comparing the differential abundance ofproteins between benign and tumorous tissue from 31 prostate cancerpatiens, we identified annexin A3 as more being variously differentiallyabundant in tumors and potentially represented a diagnostic marker forvarious sub-types of prostate cancer. Annexin A3 is a relativelyinfrequent annexin family member that was upregulated an average of 2.4fold across all 31 patients (between 1.1 and 5.4 fold with 95%confidence; P=0.045). In a tentative sub-cluster of 22 patients that wassuggested by cluster analysis, Annexin A3 was upregulated an average of4.4 fold (between 2.2 and 9.1 fold with 95% confidence; P=0.0008),suggesting that in certain types of tumor Annexin A3 abundance may beinvolved in the cancerous phenotype. For further details it is referredto PCT/EP2005/001567, the subject matter of which is incorporated hereinin its entirety.

Several annexins are reported to be down-regulated in association withprostate cancer, including annexin A1, annexin A2, annexin A4, annexinA7 and annexin A10 (6). Alaiya et al. (18) also reported “somedifferential (annexin A3) value between malignant and benign” prostatetissue. Recently, annexin A3 has been shown to be necessary for DNAreplication in cultured hepatocytes (19), and seems to be expressedhigher in small hepatocytes which have higher growth potential andproliferation rates than parenchymal hepatocytes (20). We thus believethat annexin A3, typically a rare member of this family, may thereforeprovide a biomarker or target or therapeutic principle for cancertreatment of certain patients.

Annexins are cytoplasmic, but are also found extracellularly, althoughthey lack secretory leader sequences. For instance, Carlsson et al. (24)identified annexin A3 as an antigen for anti-sperm antibodies involvedin male infertility. Oh et al. (25) found that annexin A1 was exposed onepithelial surfaces in the vicinity of solid lung tumors, and thatadministration of a radiolabelled antibody against the protein causedtumor regression in animal experiments. Indeed, annexin A5 translocationto cell surfaces is associated with apoptosis (26), and annexin A1, alsoknown as Lipocortin 1, is released to the extracellular space in largeabundances from neutrophils and monocytes/macrophages as ananti-inflammatory agent. In fact, annexin A1 may be the primary mediatorof the anti-inflammatory effect of glucocorticoids (27, 28).

There is no mechanism for annexin secretion reported (17) combiningsecretion, especially the cellular expulsion of annexin A3, the exosomepathway, and altered regulation of immune surveillance of the prostate.Exosomes are membrane vesicles of 30 to 100 nm in diameter, which areproduced and secreted in vitro by living cells of diverse origin, andare thought to be involved in the transfer of tumor antigens to antigenpresenting cells, as well as in the stimulation of specific immuneresponses (21). Annexin family members, including annexin A3 and annexinA8, are commonly found in exosomes (21-23).

Hegmann et al. (29) have postulated that exosomes are involved in therelease of heat shock proteins to the extracellular environment in theabsence of cellular necrosis. The luminal exosome environment couldpermit the low pH values necessary for the proposed annexin calcium ionchannel function in vivo, that has been controversially discussedbecause of incompatibility with cell viability (17). Indeed, thereported instances of physiological annexin ion channels occur in thematrix vesicles involved in osteoblast bone formation, and in theterminal differentiation and death of chondrocytes (30), which are bothcircumstances atypical of normal cellular viability. We thus believethat annexin ion channels could be involved in the osmotic rupture ofexosome vesicles (either within multivesicular vesicles prior tosecretory fusion with the cytoplasmic membrane, or extracellularly) andthereby modulate extracellular milieu of tumors or other tissues, suchas bone in the case of osteoporosis.

Bondanza et al. (31) recently reported that irradiated tumor cells areefficiently phagocytized by macrophages, but when cell surfacephosphatidylserine is masked by annexin A5, the macrophage pathway isreduced and a strong CD8+ dendritic cell-dependent immune response iselicited. As referred to above, annexin A1 is an anti-inflammatorymodulator that reduces neutrophil recruitment, and thereby reducestissue inflammation. It binds to specific extracellular ALX (lipoxin A)receptors on neutrophils and macrophages, and can thereby modulatemacrophage phagocytosis (27, 28). At the site of action within thetissues, annexin A1 and its N-terminal peptide (Ac2-26) promotesphagocytosis of apoptotic neutrophils, thereby reducing the level ofinflammation and the immune response through anti-inflammatory cytokinessuch as TGF (Transforming Growth Factor)-β1 (28), and, accordingly, theantigen-induced T cell proliferation of Th1 (T-helper 1) and Th2(T-helper 2) T-cells is also inhibited by the peptide Act-26 (32).Changes of annexin A3 in tumors may influence immune surveillance ofprostate tissue by altering the properties and/or concentration of theextracellular annexin pool, and by thereby modulating the interplaybetween a macrophage/granulocyte dominated response, and/or a humoralone.

The abundance of at least one annexin protein may be determined togetherwith the abundance of at least another annexin protein, preferably ofthe group consisting of annexin A1, annexin A2, annexin A3, annexin A4,annexin A5, annexin A6, annexin A7, annexin A8 and annexin A10.

The abundance of at least one annexin protein may also be determinedtogether with the abundance of at least a further protein of the groupconsisting of serum amyloid P, isopeptidase T, muscle-type fatty acidbinding protein, galectin 1, heat shock protein 90, BiP (Human protein:P11021-78 kDa glucose-regulated protein precursor, GRP 78,Immunoglobulin heavy chain binding protein, Endoplasmic reticulumlumenal Ca2+ binding protein grp78), protein disulfide isomerase,epidermal-type fatty acid binding protein, enoyl coenzyme A hydrataseand nucleophosmin.

Furthermore, the abundance of at least one annexin protein can bedetermined together with the abundance of at least a further protein ofthe group consisting of 14-3-3 family, proteasome, particularly prosomeand/or macropain, activator subunit 2, cytokeratin family, KNP-I alphaprotein (NCBI ACCESSION BAA95554.1 GI:7768772) and KNP-1 beta protein(NCBI ACCESSION BAA21139.1 GI:2250701).

In some cases the diagnostic value of conventional tumor markers fordiagnosis is limited. For instance, high or extremely low serum prostateantigen (PSA) values provide a reasonable reliable diagnostic index forprostate cancer. However, preoperative PSA values ranging between 2 and10 ng/ml, especially between 4 and 10 ng/ml, particularly between 2 and6 ng/ml, are extremely poor regarding diagnostic reliability, inparticular with respect to prediction of postoperative cure rates inradical prostatectomies.

Thus, in a particular preferred aspect the abundance of at least oneannexin protein is determined together with the abundance of at leastone blood or serum marker, in particular of at least one member of theKallikrein protease family, preferably of prostate specific antigen(PSA). The abundance of various forms of PSA, in particular total PSA(tPSA) abundances, relative or absolute abundances of free PSA (fPSA)and relative or absolute abundances of complexed PSA (cPSA), may bedetermined together with the abundance of annexin A3. It is furtherwithin the scope of this disclosure that other members of the Kallikreinprotease family may be used in this respect. The abundances of theseproteins to one another may also be used in combination with one or moremeasured or calculated annexin parameters for diagnostic purposes. Theannexin parameters that can be useful are obviously not restricted tothose used by way of demonstration in this disclosure.

The abundance of at least one annexin protein may also be determinedtogether with the abundance of at least an epithelial cell marker,particularly prostate specific membrane antigen (PSMA).

According to an especially preferred aspect, annexin A3 and/or annexinA8, preferably annexin A3, are used.

The cancer to be treated and/or diagnosed can be derived from theurogenital and/or intestinal tract. Preferably, cancer is chosen fromthe group consisting of prostate cancer, kidney cancer, bladder cancer,urethra cancer, ovarian cancer, uterine cancer or colon cancer.Preferably, the cancer to be diagnosed is prostate cancer and/or coloncancer. With respect to prostate cancer, the method preferably allowsfor discrimination between prostate cancer tissue samples, benignprostatic hyperplasia (BPH) tissue samples, chronic prostatitis tissuesamples, fibrosis afflicted tissue samples and healthy tissue samples.

Concerning cancer of the intestinal tract, particularly colon cancer,the method preferably allows for discrimination between cancer tissuesample and samples of tissue which are affected by inflammatory bowldiseases, particularly Crohns's disease and/or colitis ulcerosa.

It is possible to treat and/or to diagnose subgroups of cancers.Furthermore, different cancer stages may be treated and/or diagnosed. Itis further possible to monitor the transition of non-cancerous tissueinto cancerous tissue.

In a further preferred aspect, excrement samples or fractions thereof,especially of urine, in particular of exprimate urine, are subjected toa separation process prior to determining the abundance of at least oneannexin protein to yield cell pellets and supernatants. Preferably, theseparation process is done by centrifugation, especially by low speedcentrifugation of cells out of a liquid medium (e.g., 200×g for 5minutes at 4° C.). Any suitable centrifugation protocol, includingsuccessive centrifugations under different conditions, or combinationsof centrifugations with other methods, may be employed to separatesoluble or exosome-bound annexin from intracellular annexin formeasurement. Other means of separation of soluble or exosome-boundannexin from intracellular annexin can also be employed, or combinationsthereof (e.g., magnetic beads, filtration, chromatography, etc).

The cell pellets may be used for determining the intracellular abundanceof at least one annexin protein, preferably of annexin A3.

As already mentioned in the above description, annexins are intricatelyinvolved in processes of osteoblastosis and osteolysis. Annexins areimplicated in the process of bone mineralization. This is noteworthybecause prostate cancer metastases are unusual among cancers inexhibiting a high frequency of osteoblastic bone lesions. Most cancermetastases are characterized by osteoclast osteolytic (bone dissolving)activity, whereas prostate metastases exhibit both osteoclastic andmineral depositing osteoblastic activity.

Physiological mineralization is a highly complex and regulated process.Bone mineralization is initiated by small vesicles, called matrixvesicles, that are released from the plasma membrane of mineralizingskeletal cells. The first mineral phase forms inside the matrixvesicles. Since these are membrane-enclosed, channel proteins arerequired for the mineral ions to enter. Annexins form channels into thematrix vesicles by which Ca²⁺ enters, leading to the initiation ofcalcium phosphate minera-lization. Once the intravesicular crystalsreach a certain size they rupture the membrane. This is in turn relatedto inflammation, a feature common to cancer and annexin biology, andinvolves an interplay between bone and the immune system. Therefore, themethod can be used to diagnose and/or treat osteoporosis. The methodannexin abundances, preferably the abundance of annexin A3 and/orannexin A8, may be determined in body fluids, body secretions, tissuesamples, groups of cells or cells, especially by methods known to thoseskilled in the art to diagnose and/or treat osteoporosis. Such treatmentmay involve the application of substances that influence the abundance,subcellular/extracellular localisation, post-translational modificationor activity of annexin proteins. Activity in this respect especiallyincludes ion-channel activity, which may be appropriately increased ordecreased. Substances that may be used for the treatment of osteoporosisexplicitly include annexin A3, truncated or mutant versions thereof, orantibodies or other affinity reagents. The substances can furtherinclude nucleic acids, or chemically related substances, such as peptidenucleic acids (pNA), which may be also used a small interfering RNAs(siRNAs) as known in the art.

Examples of protein analysis of exprimate massaged prostate urine cellpellets from patients diagnosed with cancer, benign prostatichyperplasia (BPH) or control patients with conditions diagnosed asunrelated to cancer are shown in FIGS. 3-5, respectively. The top panelsin each of FIG. 3 to FIG. 5 show annexin A3 enhanced chemiluminescence(ECL) signal from western blot, and the bottom panels show the entireloaded protein signal as stained with Ponceau S (‘Protein’). Each gelcontains a molecular weight ladder (M) as well as duplicates of 7.5 μgof total cell protein lysate from a prostate tumor containing annexin A3as a positive control (+C). Annexin A3 signals from samples on differentgels can be compared by normalization to the average value of thereplicate respective positive controls. We found in a preliminary studythat the pellets of exprimate urine samples of cancer patients have muchless annexin A3 than either benign prostatic hyperplasia (BPH) patientsor healthy control patients (FIG. 6). For instance, regarding thereference value of 0.2-fold the amount of annexin A3 signal (abundance)as in PR_(—)26CA, only 5/30 (5:25) exprimate urine samples of cancerpatients have more than 0.2 fold the reference value, while 23/30 (23:7)of the exprimate urine samples of BPH patients and 18/30 (18:12) of theexprimate urine samples of healthy control patients exceeded thisreference value. These results show that on average, those cell pelletsfrom samples of exprimate urine of cancer patients contain less annexinA3 than samples of exprimate urine from BPH patients or healthy controlpatients.

The supernatants resulting from the separation process of the urinesamples, in particular of exprimate urine samples, and fractions thereofmay be used to determine the extracellular abundance of at least oneannexin protein, preferably of annexin A3.

It is particularly preferred to use the supernatants for diagnosingcancer, in particular, of the urogenital and/or intestinal tract and/orfor discrimination between cancerous and non-cancerous tissue.

A cation chelator, especially a Ca²⁺-chelator, particularly EDTA and/orEGTA, may be added to the urine sample or fraction thereof prior todetermining the abundance of at least one annexin protein, preferably ofannexin A3. The addition of the cation chelator may occur prior tosubjection of the samples or fractions thereof to the separatingprocess.

Preferably, determination of the abundance of at least one extracellularannexin protein, particularly annexin of A3, is performed in an cationchelator treated, particularly EDTA and/or EGTA treated, supernatant andfor comparison in a supernatant lacking a cation chelator, particularlyEDTA and/or EGTA. The supernatant is preferably derived from the sameurine sample, in particular exprimate urine sample, or a fractionthereof.

Based on the reasoning that in particular annexin A3 translocation fromthe interior to the exterior of cells is differentially affected duringthe development of prostate cancer, we include the determination ofwhether there is any difference in the intracellular/extracellularlocalisation of annexin A3 in assocation with cancer. The extracellularenvironment as already mentioned is understood as the extracellularspace including the outer surface of plasma membranes of cells.Exprimate urine obtained subsequent to clinical prostate massagecontains cells exuded from the prostate. As well as the possibility ofextracellular annexin A3 in exosomes, free annexin A3 could bind tonegatively charged groups such as phospholipids on the surface of cellsin a calcium-dependent manner. The latter annexin A3-fraction could bereleased from the surface of cells into the supernatant by addition ofEDTA/EGTA to the medium to chelate calcium.

A further investigation in a double-blinded, four-center studydemonstrated that the ratio of total annexin A3 of pellet oversupernatant was able to diagnose cases labelled fibrosis in the group ofnon-cancer patients. Fibrosis is associated with benign processes andindicative of non-cancer. The AUROC was 0.7072 for ‘pu.anx.tot.ratio’for a total of 103 non-cancer cases. The correlation for the ratio wasnegative, thus increased total annexin A3 amounts in supernatants werecrucial for assorting into this group. This is logic as for cancer casesa decreased annexin A3 value in supernatants was observed (see below).

The further profiling of non-cancer patients (BPH, chronic prostatitis,fibrosis, PIN1-3) by ratios of annexin A3 in pellets/supernatants is animportant aspect for subsequent sequential and/or multiparameter stepsof data analysis beyond diagnostic decision cancer vs. non-cancer.

Additionally, we determined annexin 3 abundances in supernatants andcell pellets of a separate independent series of patients, to comparethe relative annexin A3 abundance in both cell pellets and supernatantsof exprimate urine samples. Again, it was found for this differentpatient cohort that abundances of annexin A3 in samples of exprimateurine pellets from cancer patients are lower than abundances of annexinA3 in samples of exprimate urine pellets from BPH patients or healthypatients. Concerning the supernatants, the annexin A3 abundances fromthese same patients are higher in the EDTA-treated supernatants ofexprimate urine samples of cancer patients than in EDTA-treatedsupernatants of exprimate urine samples of BPH patients or healthypatients. From individual ratios of extracellular (EDTA-treatedsupernatants) and intracellular (1000×g pellets) fractions of exprimateurine an even clearer image emerged, as shown in FIG. 7. Taken together,these data indicate false positive rates around or below 10% andmoreover the ratios of annexin A3 expression in supernatants (annexinA3-S) vs. pellets (annexin A3-P) allows a discrimination of cancer vs.BPH vs. controls as shown in Table 1: essentially annexin A3-S is highin cancer and BPH, and low in controls, whereas annexin A3-P is high inBPH and controls and low in cancer; thus having high S (or S/P), low Pfor cancer; high S (or S/P) and high P for BPH; and low S (or S/P) andhigh P for controls; individual ratios (S/P) give clearest picture ascompared to pellets alone (FIG. 7). Additional calibration for proteinabundances further improves the picture.

The abundance of at least one annexin protein, preferably annexin A3,may be determined by immunohistochemical methods, in particular usingtissue samples, such as tissue sections.

We also use at least one anti-annexin antibody, in particular ofanti-annexin A3 antibody, for diagnosis of cancer, in particular of theurogenital and/or intestinal tract, and/or for discrimination betweencancerous and non-cancerous tissue. The anti-annexin antibody may beused for pathohistological-diagnostically staining of tissue samples, inparticular tissue sections. The samples may be obtained by biopsies orcomplete tissue excision. In particular, the tissue samples to bestained by the anti-annexin antibody are derived from prostate biopsiesor prostate tissue after protectomy.

A polyclonal rabbit serum containing antibodies against annexin A3 wasobtained and used to localize annexin A3 in prostate tissues by immunehistochemistry. Because of the large number of annexin family members,we characterized the specificity of the anti-annexin A3 polyclonalantibody by Western blot prior to immune histochemistry. The vastmajority of the signal obtained by Western blot of prostate benign andcancer tissue cell lysates comes from annexin A3 (FIG. 1). A marginalabundance of signal was observed for higher molecular weight protein,which is presumably annexin A6. This antibody produced a strong cleanband using approximately 120 ng recombinant 60 kDa GST(Glutathione-S-Transferase)-annexin A3 under the same conditions. Athorough quantification, based on radioactive values from 2D gels frombiopsies, protein stains of 1D and 2D gels and 1D and 2D Western blotsfrom biopsies and exprimate urines, lead to determination of detectionlimits of protein concentrations in exprimate urine samples ranging from0.02 to >15 ng/ml. The limit of detection was somewhere below but nearto 0.01 ng/ml. In terms of protein content the range is from 0.001 tomore than 0.3 ng/μg total protein.

It is therefore probable that this antibody recognizes predominantlyannexin A3 in immune histochemistry, as shown in FIG. 2, where thecorresponding annexin A3 signal is restricted to epithelial cells inhealthy prostate, and additionally to cancerous cells in tumors. Stromalcells exhibited enhanced staining in early cancer. The rationale ofmechanism for differences of annexin A3 in prostate cancer and BPHtissue tentatively indicates a transition from intracellular, morelocalized and in total lower expression to extracellular and in totalhigher expression when compared to controls.

The distribution of annexin A3 staining was suggestive of cytoplasmicand membrane localisation (FIG. 2), although the overall level ofstaining in individual cells seemed lower in cancer than benign tissue(FIG. 2), the overall level of annexin A3 in the same cancer tissue washigher (e.g., FIG. 1), which may be explained by more annexinA3-containing cells in cancerous tissue, and/or more extracellularannexin A3 in cancerous tissue.

In a comprehensive study (four centers, double-blinded) taking intoaccount and measuring for the first time total annexin amounts insupernatants and pellets of exprimate urine of 250 patients and moreoverquantifying the potential contribution of neutrophils to the annexin A3signal (by parallel quantification of neutrophil marker NGAL), it wasobserved that annexin A3 levels were reduced in the supernatants ofexprimate urine of patients with cancer. In general terms, this resultindicates, that higher levels of annexin A3 are observed in exprimateurine of non-cancer patients with fibrosis/BPH, than in cancer patients.However, these levels were much reduced to negligible in non-exprimateconventional urine. Therefore, it is concluded that the annexin A3measured in exprimate urine originates predominantly in the prostate,and is released into the urine as a consequence of the prostatic massageprocess. It has been shown above that annexin A3 is expressed primarilyin the ductal epithelial cells of the healthy prostate. Fordiscrimination of cancer vs. non-cancer alone, the data indicate thatamount of annexin A3 in supernatants has the biggest diagnostic value(AUROC-values for a combined readout of annexin A3 per μg protein andtotal annexin A3 in supernatants of exprimate urine of prostate cancerpatients with initial PSA-values between 4-10 were 0.78-0.82). It wasobserved a disturbance of pellet-annexin A3 in case of higherNGAL-values; so corresponding AUROC's for pellet annexin A3 were in therange of 0.55-0.65).

It is also known that in a prostate with cancer, only a small percentageof the epithelial ductal cells are cancerous. Therefore, the measureddifferences in extra-cellular annexin A3 abundance should benon-significant according to logical reasoning. Nevertheless, aconsiderable and significant reduction of average annexin A3 abundancein the exprimate urine of cancer patients relative to non-cancerouspatients was observed. It is not possible to rationalize why this shouldbe the case because the non-cancerous epithelial cells should continueto secrete annexin A3 according to intuitive conventional wisdom.Possibly, the presence of cancer causes the secretion of a trans-actingsubstance, such as cytokine, that affects the annexin A3 secretion fromthe bulk of epithelial cell in a prostate with a cancerous lesion. It isunclear whether this trans-acting factor originates in the cancerouscells themselves, or in other cells. It is well documented thattrans-acting factors influence the relationship between cancerous celland their mesenchymal/stromal environment, and vice versa. Irrespectiveof the mechanism(s) responsible, our empirical observations areunambiguous, and clearly but surprisingly demonstrate that lower levelsof annexin A3 in the exprimate urine provide a predictive measure of theprobability that the patient has tumorous cells in the prostate. Thisdiagnostic use of annexin A3 levels in exprimate urine can be combinedwith other diagnostic indexes, such as the level of prostate specificantigen (PSA), as demonstrated by way of example. These results alsosuggest that the presence of the annexin A3 protein is associated withthe healthy phenotype. Therefore, annexin A3 protein can be applied in atherapeutic manner to treat cancer, by enhancing the levels ofextracellular annexin A3.

The mechanisms underlying the observed results are under investigation,and they potentially reflect a transition of some kind of completelyhealthy prostate epithelium proceeding to a non-cancerous stage(fibrosis/BPH) which is associated with elevated levels of annexin A3 inboth pellets and supernatants of exprimate urine, with the above ratioof total annexin A3 (p/s) with highest diagnostic value. In cancer,there is a clear and surprising correlation to decreased annexin A3amounts in supernatants of exprimate urine of cancer patients; pelletannexin A3 amounts appear to have a contaminating contribution byNGAL-positive leukocytes/neutrophils.

It is thus highly desirable to measure annexin A3 levels in both pelletand supernatant. This information on the protein level is not accessibleby genomic methods e.g., as superficially insinuated but notsubstantiated by US2003/0108963A1.

In summary, annexin A3 exhibited predominantly intracellular staining inhealthy tissue, and extra-epithelial location in early cancerous tissue:with advanced cancer exhibiting markedly reduced annexin A3 stainingwithin cancer cells.

The urine samples or fractions thereof may be obtained from urine, inparticular from exprimate urine, which is recovered subsequent toprostate massage, particularly by rectal finger insertion.

The urine samples or fractions thereof may be purified, particularly arefreed from neutrophils, monocytes or peripheral blood mononuclear cells(PMBCs), especially by means of magneto beads. Preferably, samples orfractions thereof of morning urine are used.

Annexin levels may be measured in faeces or epithelial cells of theintestinal tract. Further, annexin levels can be used to treat and/or todiagnose epithelial cancers of gastrointestinal tract in any fraction orpreparation of faeces (any potentially exosome-producing epithelialsurface). Annexin levels are used further to treat and/or to diagnosecolorectal cancer. The methods can be combined with a determination ofneutrophils, in particular of calprotectin and/or neutrophilgelatinase-associated lipocalin (NGAL), to discriminate inflammatoryconditions (Crohn's disease or colitis ulcerosa from cancer).

At least one annexin protein, preferably annexin A3 and/or annexin A8,in particular annexin A3, can be used as diagnostic marker and/ortherapeutic target for diseases disclosed in the description, inparticular for prostate cancer, colorectal cancer and/or osteoporosis,preferably for subgroups thereof.

It is possible to treat cancer, in particular, of the urogenital and/orintestinal tract. This is preferably achieved by the enhancement of thein vivo abundance of at least one annexin protein, for instance of atleast one extracellular annexin.

Furthermore, we can diagnose cancer, in particular of the urogenitaland/or intestinal tract, and/or the discrimination between cancerous andnon-cancerous tissue. This is particularly achieved by the determinationof the ratios of intra-versus extracellular abundances and extra-versusintracellular abundances respectively of annexin proteins which may be,if appropriate, combined with the determination of the correspondingratios for other proteins. Preferably, diagnosis of cancer and/ordiscrimination between cancerous and non-cancerous tissue is based onthe extracellular abundance of at least one annexin protein. Thedetermined protein ratios and abundances respectively reveal differencesbetween cancerous and non-cancerous tissues, thus allowing for apatient's profiling. Therefore, annexin proteins, in particular annexinA3, are reliable diagnostic markers that may even completely substitutetumor markers that are conventionally applied in cancer diagnosis.

For a more detailed description, reference will now be made to theaccompanying tables and figures:

TABLE 1 # of samples Cancer BPH Controls False neg False pos 40 40 40 (%of 120) (% of 120) ANXA3-P (% of 40) Biomarker down 84 23 40 6 13.3Biomarker up 16 77 60 ANXA3-S (% of 40) Biomarker down 5 20 91 Biomarkerup 95 80 9 Ratio ANXA3-S/ ANXA3-P and sorting Cancer 92 12 3 1.5 7.5 BPH6 80 6 Control 2 8 91 Table 1: Summary of diagnostic results, annexin A3abundances are low in pellets (annexin A3-P) of exprimate urine samplesof cancer patients and comparatively high in corresponding pellets ofexprimate urine samples of BPH patients and healthy patients. Forannexin A3 abundances in supernatants (annexin A3-S) of exprimate urinesamples, there is a different picture: they are low for healthy patientsand high for cancer and BPH patients. The combined read-out correctlyassorts the three cases with numbers indicated in the lower part of thetable. Concerning further details it is referred to the abovespecification.

TABLE 2 Table 2: Protein parameters measured from exprimate urinesupernatant and pellets fractions. Name Description P_ug_tot Pellet:Total Protein amount (μg) U_ug_tot Supernatant*: Total Protein amount(μg P_ANX_ug Pellet: Annexin signal level per μg protein P_ANX_totPellet: Annexin signal level per total patient sample U_ANX_ugSupernatant: Annexin signal level per μg protein U_ANX_tot Supernatant:Annexin signal level per total patient sample PU_ANX_ug Pellet +Supernatant: Annexin-level per μg protein PU_ANX_tot Pellet +Supernatant: Annexin-level per total patient sample PU_ANX_ug_ratioRatio of Pellet/Supernatant: Annexin level per ug proteinPU_ANX_tot_ratio Ratio Pellet/Supernatant: Annexin level per totalpatient sample P_NGAL_ug Pellet: NGAL signal level per μg proteinP_NGAL_tot Pellet: NGAL signal level per total patient sample U_NGAL_ugSupernatant: NGAL signal level per μg protein U_NGAL_tot Supernatant:NGAL signal level per total patient sample PU_NGAL_ug Pellet +Supernatant: NGAL-level per μg protein PU_NGAL_tot Pellet + Supernatant:NGAL-level per total patient sample PU_NGAL_ug_ratio Ratio ofPellet/Supernatant: NGAL level per ug protein PU_NGAL_tot_ratio RatioPellet/Supernatant: NGAL level per total patient sampleP_ANX_NGAL_ug_ratio Pellet: Annexin/NGAL ratio per μg proteinU_ANX_NGAL_ug_ratio Supernatant: Annexin/NGAL ratio per μg proteinPU_ANX_NGAL_ug_ratio Pellet + Supernatant: Annexin/NGAL ratio per μgprotein (‘U’ = ‘supernatant’)

TABLE 3 Table 3: description of abbreviations and variables used inexample 4. Variable Description P.ug.tot Pellet: Total Protein Quantity(μg) U.ug.tot Supernatant: Total Protein Quantity (μg) P.ANX.ug Pellet:Annexin-Level per μg Protein P.ANX.tot Pellet: Annexin-Level per totalSample U.ANX.ug Supernatant: Annexin-Level per μg Protein U.ANX.totSupernatant: Annexin-Level per total Sample PU.ANX.ug Supernatant +Pellet: Annexin-Level per μg Protein PU.ANX.tot Supernatant + Pellet:Annexin-Level per total Sample PU.ANX.ug.ratio Ratio Pellet/Supernatant:Annexin Level per ug Protein PU.ANX.tot.ratio Ratio Pellet/Supernatant:Annexin Level per total Sample psa.ini Blood PSA levels perc.free.psaPercentage free PSA

TABLE 4 Table 4: ROC curve analysis results for the indicated variableparameters (see Table 3 for description), performed for patients groupedaccording to PSA values of 4-10 ng/mL. Test AUROC Patients anx.comb.var0.78 112 comb.var.anx.psa 1 0.76 112 u.anx.tot 0.76 112 pu.anx.tot 0.75112 u.anx.ug 0.74 112 perc.free.psa 0.72 103 psa.ini 0.57 109 The testparameters, resulting AUROC values, and number of patients included peranalysis are tabularized.

TABLE 5 Table 5: ROC curve analysis results for the indicated variableparameters (see Table 3 for description), performed for patients groupedaccording to PSA values 2-6 ng/mL. Test AUROC Patients comb.var.anx.psa2 0.79 109 comb.var.anx.psa 1 0.77 109 pu.anx.tot 0.73 109 anx.comb.var0.71 109 u.anx.tot 0.71 109 pu.anx.ug 0.71 109 u.anx. 0.71 109 p.anx.tot0.69 109 psa.ini 0.69 109 perc.free.psa 0.69 109 The test parameters,resulting AUROC values, and number of patients included per analysis aretabularized.

TABLE 6 Table 6: ROC curve analysis results for the indicated variableparameters (see Table 3 for description), performed for all patients,including all PSA values. Test AUROC Patients comb.var.anx.psa 1 0.75226 comb.var.anx.psa 2 0.73 244 perc.free.psa 0.70 227 psa.ini 0.68 239anx.comb.var 0.67 244 pu.anx.tot 0.66 245 The test parameters, resultingAUROC values, and number of patients included per analysis aretabularized.

TABLE 7 Criterion Sens. (95% C.I.) Spec. (95% C.I.) +LR −LR +PV−PV >=−3.5155 100.0 (93.7-100.0) 0.0 (0.0-6.9) 1.00 52.3 >−3.5155 98.2(90.6-99.7) 0.0 (0.0-6.9) 0.98 51.9 0.0 >−2.6907 98.2 (90.6-99.7) 1.9(0.3-10.3) 1.00 0.91 52.3 50.0 >−2.5444 98.2 (90.6-99.7) 3.8 (0.6-13.2)1.02 0.46 52.8 66.7 >−2.0663 98.2 (90.6-99.7) 5.8 (1.3-16.0) 1.04 0.3053.3 75.0 >−1.9579 98.2 (90.6-99.7) 7.7 (2.2-18.6) 1.06 0.23 53.880.0 >−1.9096 98.2 (90.6-99.7) 9.6 (3.2-21.0) 1.09 6.18 54.483.3 >−1.6827 98.2 (90.6-99.7) 11.5 (4.4-23.5) 1.11 0.15 54.985.7 >−1.6673 98.2 (90.6-99.7) 13.5 (5.6-25.8) 1.14 0.13 55.487.5 >−1.5429 96.5 (87.9-99.5) 13.5 (5.6-25.8) 1.12 0.26 55.077.8 >−1.4419 96.5 (87.9-99.5) 15.4 (6.9-28.1) 1.14 0.23 55.680.0 >−1.3903 94.7 (85.4-98.8) 15.4 (6.9-28.1) 1.12 0.34 55.172.7 >−1.273 94.7 (85.4-98.8) 17.3 (8.3-30.3) 1.15 0.30 55.775.0 >−1.2619 94.7 (85.4-98.8) 19.2 (9.6-32.5) 1.17 0.27 56.276.9 >−1.1825 93.0 (83.0-98.0) 19.2 (9.6-32.5) 1.15 0.36 55.871.4 >−1.1791 93.0 (83.0-98.0) 21.2 (11.1-34.7) 1.18 0.33 56.473.3 >−1.0689 91.2 (80.7-97.1) 21.2 (11.1-34.7) 1.16 0.41 55.968.7 >−1.0621 91.2 (80.7-97.1) 23.1 (12.5-36.8) 1.19 0.38 56.570.6 >−1.0192 91.2 (80.7-97.1) 25.0 (14.0-38.9) 1.22 0.35 57.172.2 >−1.0041 91.2 (80.7-97.1) 26.9 (15.6-41.0) 1.25 0.33 57.873.7 >−0.9502 91.2 (80.7-97.1) 28.8 (17.1-43.1) 1.28 0.30 58.475.0 >−0.8458 89.5 (78.5-96.0) 28.8 (17.1-43.1) 1.26 0.36 58.071.4 >−0.8339 89.5 (78.5-96.0) 30.8 (18.7-45.1) 1.29 0.34 58.672.7 >−0.8285 87.7 (76.3-94.9) 30.8 (18.7-45.1) 1.27 0.40 58.169.6 >−0.8253 87.7 (76.3-94.9) 32.7 (20.3-47.1) 1.30 0.38 58.870.8 >−0.8237 87.7 (76.3-94.9) 34.6 (22.0-49.1) 1.34 0.35 59.572.0 >−0.8044 87.7 (76.3-94.9) 36.5 (23.6-51.0) 1.38 0.34 60.273.1 >−0.7935 87.7 (76.3-94.9) 38.5 (25.3-53.0) 1.43 0.32 61.074.1 >−0.7836 87.7 (76.3-94.9) 40.4 (27.0-54.9) 1.47 0.30 61.775.0 >−0.7431 87.7 (76.3-94.9) 42.3 (28.7-56.8) 1.52 0.29 62.575.9 >−0.7371 86.0 (74.2-93.7) 42.3 (28.7-56.8) 1.49 0.33 62.073.3 >−0.6841 86.0 (74.2-93.7) 44.2 (30.5-58.7) 1.54 0.32 62.874.2 >−0.6759 86.0 (74.2-93.7) 46.2 (32.2-60.5) 1.60 0.30 63.675.0 >−0.6297 86.0 (74.2-93.7) 48.1 (34.0-62.4) 1.66 0.29 64.575.8 >−0.6205 86.0 (74.2-93.7) 50.0 (35.8-64.2) 1.72 0.28 65.376.5 >−0.6095 86.0 (74.2-93.7) 51.9 (37.6-66.0) 1.79 0.27 66.277.1 >−0.5341 86.0 (74.2-93.7) 53.8 (39.5-67.8) 1.86 0.26 67.177.8 >−0.5077 86.0 (74.2-93.7) 55.8 (41.3-69.5) 1.94 0.25 68.178.4 >−0.503 86.0 (74.2-93.7) 57.7 (43.2-71.3) 2.03 0.24 69.078.9 >−0.4942 86.0 (74.2-93.7) 59.6 (45.1-73.0) 2.13 0.24 70.079.5 >−0.4548 84.2 (72.1-92.5) 59.6 (45.1-73.0) 2.09 0.26 69.677.5 >−0.4346 82.5 (70.1-91.2) 59.6 (45.1-73.0) 2.04 0.29 69.175.6 >−0.4211 80.7 (68.1-89.9) 59.6 (45.1-73.0) 2.00 0.32 68.773.8 >−0.3763 78.9 (66.1-88.6) 59.6 (45.1-73.0) 1.95 0.35 68.272.1 >−0.201 78.9 (66.1-88.6) 61.5 (47.0-74.7) 2.05 0.34 69.272.7 >−0.1764 78.9 (66.1-88.6) 63.5 (49.0-76.4) 2.16 0.33 70.373.3 >−0.1476 78.9 (66.1-88.6) 65.4 (50.9-78.0) 2.28 0.32 71.473.9 >−0.1049 77.2 (64.2-87.2) 65.4 (50.9-78.0) 2.23 0.35 71.072.3 >−0.0809 75.4 (62.2-85.9) 65.4 (50.9-78.0) 2.18 0.38 70.570.8 >−0.0773 75.4 (62.2-85.9) 67.3 (52.9-79.7) 2.31 0.36 71.771.4 >−0.0323 73.7 (60.3-84.5) 67.3 (52.9-79.7) 2.25 0.39 71.270.0 >−0.0007 71.9 (58.5-83.0) 67.3 (52.9-79.7) 2.20 0.42 70.768.6 >0.007 71.9 (58.5-83.0) 69.2 (54.9-81.3) 2.34 0.41 71.969.2 >0.0841 70.2 (56.6-81.6) 69.2 (54.9-81.3) 2.28 0.43 71.467.9 >0.085 68.4 (54.8-80.1) 69.2 (54.9-81.3) 2.22 0.46 70.966.7 >0.1274 68.4 (54.8-80.1) 71.2 (56.9-82.9) 2.37 0.44 72.267.3 >0.1395 68.4 (54.8-80.1) 73.1 (59.0-84.4) 2.54 0.43 73.667.9 >0.1475 68.4 (54.8-80.1) 75.0 (61.1-86.0) 2.74 0.42 75.068.4 >0.1672 68.4 (54.8-80.1) 76.9 (63.2-87.5) 2.96 0.41 76.569.0 >0.1764 68.4 (54.8-80.1) 78.8 (65.3-88.9) 3.23 0.40 78.069.5 >0.1803 68.4 (54.8-80.1) 80.8 (67.5-90.4) 3.56 0.39 79.670.0 >0.1808 66.7 (52.9-78.6) 80.8 (67.5-90.4) 3.47 0.41 79.268.9 >0.2017 64.9 (51.1-77.1) 80.8 (67.5-90.4) 3.38 0.43 78.767.7 >0.2682 63.2 (49.3-75.5) 80.8 (67.5-90.4) 3.28 0.46 78.366.7 >0.2761 63.2 (49.3-75.5) 82.7 (69.7-91.7) 3.65 0.45 80.067.2 >0.3249 61.4 (47.6-74.0) 82.7 (69.7-91.7) 3.55 0.47 79.566.2 >0.3445 59.6 (45.8-72.4) 82.7 (69.7-91.7) 3.45 0.49 79.165.2 >0.4091 57.9 (44.1-70.9) 82.7 (69.7-91.7) 3.35 0.51 78.664.2 >0.476 57.9 (44.1-70.9) 84.6 (71.9-93.1) 3.76 0.50 80.564.7 >0.4824 57.9 (44.1-70.9) 86.5 (74.2-94.4) 4.30 0.49 82.565.2 >0.4969 57.9 (44.1-70.9) 88.5 (76.5-95.6) 5.02 0.48 84.665.7 >0.5666 57.9 (44.1-70.9) 90.4 (79.0-96.8) 6.02 0.47 86.866.2 >0.6639 57.9 (44.1-70.9) 92.3 (81.4-97.8) 7.53 0.46 89.266.7 >0.683 56.1 (42.4-69.3) 92.3 (81.4-97.8) 7.30 0.48 88.965.8 >0.6877 56.1 (42.4-69.3) 94.2 (84.0-98.7) 9.73 0.47 91.466.2 >0.6934 54.4 (40.7-67.6) 94.2 (84.0-98.7) 9.43 0.48 91.265.3 >0.7038* 54.4 (40.7-67.6) 96.2 (86.8-99.4) 14.14 0.47 93.965.8 >0.7226 52.6 (39.0-66.0) 96.2 (86.8-99.4) 13.68 0.49 93.864.9 >0.8531 50.9 (37.3-64.4) 96.2 (86.8-99.4) 13.23 0.51 93.564.1 >0.8868 49.1 (35.6-62.7) 96.2 (86.8-99.4) 12.77 0.53 93.363.3 >0.9511 47.4 (34.0-61.0) 96.2 (86.8-99.4) 12.32 0.55 93.162.5 >0.9527 45.6 (32.4-59.3) 96.2 (86.8-99.4) 11.86 0.57 92.961.7 >1.0734 43.9 (30.7-57.6) 96.2 (86.8-99.4) 11.40 0.58 92.661.0 >1.116 42.1 (29.1-55.9) 96.2 (86.8-99.4) 10.95 0.60 92.360.2 >1.1278 40.4 (27.6-54.2) 96.2 (86.8-99.4) 10.49 0.62 92.059.5 >1.1707 38.6 (26.0-52.4) 96.2 (86.8-99.4) 10.04 0.64 91.758.8 >1.1763 36.8 (24.5-50.7) 96.2 (86.8-99.4) 9.58 0.66 91.358.1 >1.2415 35.1 (22.9-48.9) 96.2 (86.8-99.4) 9.12 0.68 90.957.5 >1.267 33.3 (21.4-47.1) 96.2 (86.8-99.4) 8.67 0.69 90.556.8 >1.3195 31.6 (19.9-45.2) 96.2 (86.8-99.4) 8.21 0.71 90.056.2 >1.3976 29.8 (18.4-43.4) 96.2 (86.8-99.4) 7.75 0.73 89.555.6 >1.4368 28.1 (17.0-41.5) 96.2 (86.8-99.4) 7.30 0.75 88.954.9 >1.4579 26.3 (15.5-39.7) 96.2 (86.8-99.4) 6.84 0.77 88.254.3 >1.4781 24.6 (14.1-37.8) 96.2 (86.8-99.4) 6.39 0.78 87.553.8 >1.6158 22.8 (12.8-35.8) 96.2 (86.8-99.4) 5.93 6.80 86.753.2 >1.6423 21.1 (11.4-33.9) 96.2 (86.8-99.4) 5.47 0.82 85.752.6 >1.6559 19.3 (10.1-31.9) 96.2 (86.8-99.4) 5.02 0.84 84.652.1 >1.7477 17.5 (8.8-29.9) 96.2 (86.8-99.4) 4.56 0.86 83.351.5 >1.7524 17.5 (8.8-29.9) 98.1 (89.7-99.7) 9.12 0.84 90.952.0 >1.7628 17.5 (8.8-29.9) 100.0 (93.1-100.0) 0.82 100.0 52.5 >1.805415.8 (7.5-27.9) 100.0 (93.1-100.0) 0.84 100.0 52.0 >1.8153 14.0(6.3-25.8) 100.0 (93.1-100.0) 0.86 100.0 51.5 >1.8787 12.3 (5.1-23.7)100.0 (93.1-100.0) 0.88 100.0 51.0 >2.1994 10.5 (4.0-21.5) 100.0(93.1-100.0) 0.89 100.0 50.5 >2.4311 8.8 (2.9-19.3) 100.0 (93.1-100.0)0.91 100.0 50.0 >2.472 7.0 (2.0-17.0) 100.0 (93.1-100.0) 0.93 100.049.5 >2.5544 5.3 (1.2-14.6) 100.0 (93.1-100.0) 0.95 100.0 49.1 >2.55953.5 (0.5-12.1) 100.0 (93.1-100.0) 0.96 100.0 48.6 >2.8088 1.8 (0.3-9.4)100.0 (93.1-100.0) 0.98 100.0 48.1 >3.6467 0.0 (0.0-6.3) 100.0(93.1-100.0) 1.00 47.7 Sens. = Sensitivity Spec. = Specificity +LR =Positive likelihood ratio −LR = Negative likelihood ratio +PV = Positivepredictive value −PV = Negative predictive value Table 7: Datacorresponding to the ROC curve of FIG. 9. The maximum likelihoodsestimate model is described in Tables 8 and 9.

TABLE 8 Error Chi-Square Parameter DF Estimate (Standard) (Wald) Pr >ChiSq Intercept 1 −2.3860 1.7461 1.8673 0.1718 Log PU ANX 1 −0.47470.1394 11.5908 0.0007 tot Log PSA ini 1 3.2942 1.0198 10.4343 0.0012Table 8: Condensed SAS Output for the logit model which leads tocomb.var.anx.psa2: Analysis of Maximum Likelihood Estimates

TABLE 9 Table 9: Condensed SAS Output for the logit model which leads tocomb.var.anx.psa2: Odds Ratio Estimates Effect Estimate (Point)Confidence (95%) Limits log PU ANX tot 0.622 0.473 0.818 log PSA ini26.956 3.652 198.934

EXAMPLES

While our methods are described in more detail with reference toexamples, those methods are by no means restricted to the examples.

Example 1 Processing of Post-Prostatic Massage Urine

Prostatic massage exprimate urine was obtained from patients undergoingclinical examination, after screening for blood prostate specificantigen (PSA) abundances had indicated elevated risk of cancer. 47 ml ofurine obtained following vigorous prostate massage by rectal fingerinsertion was added to 3 ml 0.5 M EDTA, pH 8 precooled to 0° C., andimmediately cooled to 0° C. If urine volume was <47 ml, the volume wasmade up with ice cooled phosphate buffered saline (PBS) solution. Thecooled samples were centrifuged at 3000 rpm at 0° C. for 30 minutes tocreate a cell pellet. 1 ml aliquots of the supernatant were removed,frozen in liquid nitrogen, and stored at −80° C. until use. The cellpellets were gently resuspended in 2 ml ice cold PBS and transferred toEppendorf tubes on ice, followed by centrifugation at 12000 rpm for 5minutes at 4° C. The supernatant was removed, and the pellet frozen inliquid nitrogen and stored at −80° C. until use.

Example 2 Western Blotting

SDS-PAGE gels for Western blotting were prepared using a BioRad-Mini gelapparatus and 12% T polyacrylamide gels with 1 mm spacers and 15 wells,according to manufacturer's instructions. Anti annexin A3 (annexin A3)was the same antibody described below. Diluted 1:20,000. RecombinantGST-annexin A3 protein was purchased from Abnova Corporation (#ABV0040710002; Lot: T04G01-ANNEXINA3, 0.05 μg/μl, 61 kDa). Antibody bindingwas visualized with a goat anti-rabbit IgG (Sigma A 3937, lot #121K9151)diluted 1:1000, using the ECL detection method (Pierce) and a DIANA IIICCD camera-based chemiluminescence detector (Raytest, Straubenhardt,Germany). A rabbit polyclonal serum against recombinant bacteriallyexpressed annexin A3 exhibits primary specificity for annexin A3 andsome cross reactivity for annexin A6.

Example 3 Immune Histochemistry

Immune histochemistry was performed with 5 μm paraffin tissue sectionsemploying polyclonal anti annexin A3 serum, according to a standardHorse Radish Peroxidase immunohistochemistry protocol using the ZymedPicTure PLUS Kit (Broad Spectrum, DAB, Zymed, South San Fransisco,Calif.). After immunostaining sections were counterstained with Gill'shematoxylin solution (Sigma).

Example Clinical Study Using Exprimate Urine of 250 Patients

Annexin A3 levels were determined in the exprimate urine of clinicalpatients, diagnosed as being either positive or negative for thepresence of prostate cancer (PCa). A variety of additional paramterswere examined, such as prostate specific antigen (PSA) levels in theblood, and other variables listed below.

Sample collection was as per example 1, but without the addition of EDTAto urine. Following prostate massage the entire exprimate urine volumewas collected and recorded. A Combur-10-Test® (Roche Diagnostics Cat.No. 11 203 479) was performed immediately on an aliquot of the urine torecord specific gravity, pH, Leukocyte count, and the levels of nitrite,protein, glucose, ketones, urobilinogen, bilirubin, and erythrocytes.Urine was then centrifuged at room temperature for 15 min at 1000×g. Thecell pellet and supernatant of this supernatant were handled separately.After removal of the last traces of supernatant, the pellet wasresuspended in 1 ml ice cold phosphate buffered saline and frozen inliquid nitrogen or on frozen CO₂. Separate aliquots of 2×1.8 ml and upto 2×50 ml of supernatant were similarly frozen.

Frozen protein samples were thawed, and 1/100 volumes of 2% deoxycholatewere added, followed by vortexing then addition of 1/10 volume oftrichloroacetic acid, vortexing, and 10 minutes incubation at 0° C. Thiswas followed by centrifugation at 10000×g for 15 min at 4° C. toprecipitate proteins. The supernatant was removed, and the pellet waswashed three times with ice-cold 80% acetone by vigorously vortexing thepellet to remove remaining TCA completely, followed by recentrifugationat 10000×g as before after each wash. After the final centrifugation thesupernatant was removed and the pellet was left to air dry for 2minutes, paying attention not to completely dehydrate the pellet. Thepellets were resuspended in boiling XT-sample buffer (1× XT-Buffer: 141mM TrisBase; 106 mM Tris-HCl; 2% SDS; BPB; pH about 8.5; 50 mM DTT; 35%Glycerol).

The protein concentration of each sample was estimated by loadingdefined volumes of each sample to a one dimensional SDS-polyacrylamidegel electrophoresis (SDS-PAGE) gel (Criterion XT-precast gel: Biorad,Cat#345-0119, lot #CX070706B2), which contained a serial dilution ofcalibrated amounts of rat proteins from whole liver cell lysates and waselectrophoresed in a BioRad Criterion electrophoresis device accordingto manufacturer's instruction. The gel was stained using Sypro Rubyaccording to manufacturer's instructions. Briefly, the gels were fixed2×30 min in aqueous solution containing 50% methanol, 7% acetic acid,followed by staining overnight in Sypro Ruby solution (Molecular Probes,#S12001). Gels were washed for 30 min in 10% methanol, 7% acetic acid,and then for 2×5 min in water. Protein staining with Sypro Ruby wasquantified with a Diana III CCD-based digital imager (RaytestIsotopenmessgeräte GmbH, Straubenhard Germany: Sypro Filter, 605 nm).

The intensity of protein staining of Sypro Ruby-stained gel lanes wascompared between standard proteins and patient urine samples. Forsupernatants the whole lane was used for determination. For urine Pelletsamples only the lane area below the dominating Uromodulin band wasconsidered, resulting in a “uromodulin corrected protein concentration.”

Annexin A3 and neutrophil gelatinase-associated lipocalin (NGAL,SWISSPROT Accession P80188, a marker for Neutrophils) levels in eachsample were quantified by loading normalized protein amounts to SDS-PAGEgels as described above, whereby each gel contained three replicatelanes of 2 μg of a standardized protein extract from PC3 human prostatecancer cell line, which contained a convenient reference amount of bothannexin A3 and NGAL. Proteins from these gels were western blotted ontoPVDF (polyvinylidene fluoride) membranes according to standard methodsfor 1.5 h at 15V constant voltage and a limit of 3 mA/cm².

The non-specific protein binding sites on blotted membranes were blockedby 2 h incubation with gentle shaking in TBS (175 mM NaCl, 3.5 mM KCl,20 mM Tris, pH 7.4) containing 5% redissolved dried milk powder. Primaryantibodies were added specific for annexin A3 (1:20000 dilution,polyclonal rabbit anti-human Annexin A3) or NGAL (1:500 dilution,anti-human Lipocalin, polyclonal, from goat, R&D Systems, Nr. AF1757,lot JBH025051). After incubation at room temperature for 2 h the bufferwas removed, washed three times for 10 min with TBS, and then incubatedwith the appropriate respective second antibodies against rabbit IgG(goat anti-rabbit-IgG pre-absorbed with human IgG and mouse IgG, couplesto horse radish peroxidase. Santa Cruz, #sc-2054, lot #G2005. 1:5000dilution) or goat IgG (Anti-goat IgG, from rabbit, pre-absorbed withhuman IgG and mouse IgG, coupled to horse radish peroxidise. Santa CruzBiotechnology, #sc-2922 lot #C1405. 1:5000 dilution). Enhancedchemiluminescence (ECL) was measured afer addition of Super Signal WestDura, Pierce (0.1 ml/cm²).

Values of NGAL or annexin A3 signals were normalized to the averagesignal from each of the three reference PC3 lanes on each gel, and thenormalized annexin A3 or NGAL values were used for the statisticalanalysis. From these values, the levels of both proteins were calculatedrelative to the absolute sample volume, and also normalized to proteinconcentration. These values were calculated separately for pellet andsupernatant, as well as for the ratio of pellet:supernatant. Parametersthat were correlated to cancer and compared to PSA values are summarizedin Table 2.

Clinical parameters recorded included blood PSA levels, free total PSAlevels, and complexed PSA levels, as well as histological evaluation ofprostate tissue biopsies that were obtained following donation ofexprimate urine, during the course of a standard digital rectalexamination (DRE). In cases where high levels of serum PSA and DREindicated necessity of prostectomy, the histological evaluation wasperformed on that material without prior biopsy.

The above data parameters were included in the statistical analysis,that included also clinical data recorded in the hospital. Prostatebiopsies or prostectomy were obtained from all patients, and theclinician made a diagnosis of positive or negative for PCa based onhistological examination of the tissue. Blood PSA levels were alsoobtained according to standard clinical practices. The U.S. Food andDrug Administration (FDA) has approved the PSA test for annual screeningof prostate cancer in men of age 50 and older. PSA levels between 4 and10 ng/mL (nanograms per milliliter) are considered to be suspicious andshould be followed by rectal ultrasound imaging and, if indicated,biopsy. PSA is false positive- and false negative-prone. Biopsy-detectedprostate cancer, including high-grade cancers, is not rare among menwith PSA levels of 4.0 ng/mL or less—levels generally thought to be inthe normal range.

The dataset consists of composite data files from 250 patients. InitialPSA values were available in 243 of the 250 patients and were missingfor 7 patients. Furthermore, two of the latter seven patients and fiveother patients lacked the histological results of the biopsy (prostatecancer yes/no). Hence, all results presented here only use the data onthose 243 patients whose PCa status is known: 140 patients with positiveand 103 with negative PCa diagnosis. In FIGS. 8 and 9, the ReceiverOperating Characteristic (ROC) curves generated using the clinicalresults are presented using inverse values for all ROC curves other thanfor PSA; i.e., the areas under the ROC curves were observed to correlatewith higher PSA values in cancer patients, and with a lower averageannexin A3 signal measured for cancer patients.

The area under the ROC curve (AUROC) for PSA was 0.684, which issignificantly different from an AUROC of 0.5, but somewhat artificiallyhigh due to patient recruitment in participating clinics, (as known toevery expert in the field). However we do note that our patientcollective had an unusually high proportion of cancer patients (57%)because some of the patients had been examined by our test clinics afterhaving providing high PSA readings at other centres. The AUROC valuesfor the individual annexin A3-based variables measured are in the samerange (the maximum is attained by PU_ANX_tot, AUROC 0.666; data notshown), and also significantly different from 0.5. Thus annexin A3 couldalso be used to replace PSA entirely, because in the crucial grey zoneof PSA (2-6 ng/ml and 4-10 ng/ml), annexin 3 offers considerableadvantages (see ROC curves 0.78 and 0.791 in Tables 4-6) with highspecificities at acceptable sensitivities, and shows a similar overallperformance, considering the whole range of PSA-values.

There was no preselection for annexin A3 in our patient selection, andthere was no correlation between annexin A3 values and PSA values,indicating that annexin A3 expression/secretion and PSA entry to thebloodstream are regulated by separate mechanisms. We observed nocorrelation between either PSA or annexin A3 levels with patient age.Presumably the high proportion of cancer-positive patient, some of whomwere preselected on the basis of suspiciously high PSA values measuredat other centers, upset the expected higher abundance of PSA levels thatwould be expected with increasing age. However, as annexin A3 was notpreselected it is concluded that its levels are probably notage-related.

Particular emphasis is placed on the statistical analysis of thesubpopulation of patients with initial PSA values in the interval 2ng/mL to 6 ng/mL. Again, all seven AUROC values for the measuredindividual parameters are significantly different from 0.5, althoughonly 57 PCa patients and 52 non-PCa patients meet the PSA criteriondefining the subpopulation. In contrast to the overall results, theAUROC of PSA_ini is no longer the largest one, but is superceded by fiveof the six. AUROC values of annexin A3 based variables (P_ANX_ug beingthe only exception). The highest AUROC value, 0.735, is attained byPU_ANX_tot.

Besides the PSA range 2 ng/mL-6 ng/mL the PSA range 2.5 ng/mL-12 ng/mLis of special interest: In this subpopulation PSA_ini itself performspoorly (AUROC 0.580), while U_ANX_tot performs best (AUROC 0.693). Thus,this PSA range seems appropriate to assess the characteristics of thefollowing two-step procedure, which is presented by way of methodicaldemonstration:

-   -   In the first step patients are assigned to one of three classes        depending on their initial PSA value:        -   PSA_ini<2.5→low PCa risk,        -   PSA_ini>12→high PCa risk,        -   PSA_ini in [2.5, 12]→application of a test based on            U_ANX_tot as a second step deciding whether or not invasive            diagnostic procedures are indicated.    -   (The first two cases are referred to as the “most obvious        decision” in the caption of FIG. 8.)        -   This two-step procedure is then incorporated into a single            variable, here called twostepvar1, by defining

${{twostepvar}\; 1}:=\left\{ \begin{matrix}{100000,} & {{{if}\mspace{14mu}{PSA\_ ini}} < 2.5} \\{{{U\_ ANX}{\_ tot}},} & {{{if}\mspace{14mu}{PSA\_ ini}} \in \left\lbrack {2.5,12} \right\rbrack} \\{0,} & {{{if}\mspace{14mu}{PSA\_ ini}} > 12.}\end{matrix} \right.$

-   -   Low values of U_ANX_tot indicate an increased risk of prostate        cancer and high values a lower risk. The value 100000 is chosen        in order to ensure that it should always be greater than the        largest U_ANX_tot value actually measured. The performance of        twostepvar1 can be seen in FIG. 8. (The constancy of twostepvar1        for PSA_ini values below 2.5 and beyond 12 causes the ROC curve        to start and end with a noticeable straight line segment.) The        AUROC of 0.740 is of course highly significantly different from        0.5. Moreover, a comparison with the conventional PSA test is        enlightening: For example, the criterion “PSA_ini>4” leads—in        this analysis data set—to a sensitivity of 80.3% and a        specificity of 49.5%. The same sensitivity, 80.3%, is gained by        using the criterion “twostepvar1<450”, but now a specificity of        57.4% is achieved.

The example of FIG. 8 demonstrates the principles of stepped AUROCvalues which rely on different measurements (PSA or annexin A3 values)depending upon the PSA level. These principles, as will be obvious toexperts, are the same as used in several results presented in Tables4-6, and which provide higher AUROC values. The excellent performance ofcancer prediction of the annexin A3 variables in patients withintermediate levels of serum PSA is further evidenced by considering theannexin A3-based multiple variables for patients with intermediate PSAvalues.

According to the methods and strategies demonstrated above, ROC curveanalysis was performed for the parameters shown in Tables 3-6.Additionally to those variables, ROC curve analysis was performed usingthe following parameters generated by logical regression analysis:

-   -   anx.comb.var is based upon logical regression analysis using the        combined U_ANX_ug und U_ANX_tot as variables according to the        following relationship.        ≡4.463+2.906 log(1+U_ANX_ug)−0.790 log(1+U_ANX_tot)    -   comb.var.anx.psa1 is based upon logical regression analysis        using the combined PSA_ini und U_ANX_tot as variables according        to the following relationship.        ≡0.254+1.046 log(1+PSA_ini)−0.342 log(1+PU_ANX_tot)    -   comb.var.anx.psa2 is based upon logical regression analysis        using the combined PSA_ini und PU_ANX_tot as variables according        to the following relationship.        ≡2.386+3.294 log(1+PSA_ini)−0.475 log(1+PU_ANX_tot).

While extremely high or extremely low PSA values provide relativelyreliable assignment of cancer/non-cancer status, the results of Tables4-6 clearly demonstrate that various combinations of annexin A3-basedparameters outperform PSA for intermediate values of PSA. Thus, themeasurement of annexin A3 levels in exprimate urine supernatants orpellets, preferably including supernatant to pellet ratios, providesimproved diagnostic reliability.

To demonstrate the utility of these results, the ROC curve forcomb.var.anx.psa2 is presented in detail in FIG. 9, and Table 7. ThisROC curve is based only on patients with PSA values between 2 ng/mL and6 ng/mL, and gives a highly significant AUROC value of 0.791 despite theuse of only 109 patients in this range for the analysis. Furthermore,this ROC curve exhibits an extremely steep climb in sensitivity (truepositive fraction) relative to specificity (true negative fraction),which is quite advantageous regarding predictive value. For instance ata sensitivity level of 54% the specificity is 96% (FIG. 9, Table 7). TheROC curve for anx.comb.var performed similarly, with sensitivity of 38%having specificity of 91% (AUROC 0.78) for the PSA range 4 ng/mL to 10ng/mL. The data disclosed here demonstrate convincingly that annexin A3is a novel and powerful marker for prostate cancer, that is especiallypowerful in those patients where PSA values are the least reliable.

Taken together, the third comprehensive study which was double-blindedand multi-center, showed, that the most robust and statisticallysignificant diagnostic read-out was the annexin A3-amount insupernatants of exprimate urines after prostatic massage which have beenobtained during a standard clinical procedure (DRE) due for potentialprostate cancer patients with a standard low speed centrifugation. Thisis very favorable because it allows direct access to ELISA-based orother antibody-based assays without prior solubilization of pelletedsamples (danger of interference by detergents, salts, chemicals etc.).This annexin A3-amount in supernatants is inversely correlated withcancer, in non-cancers, annexin A3-amounts are higher, with certainindications that additional and sequential profiling of non-cancer casescap even improve the overall diagnostic value.

The results are completely in line with the first two studies, whichwere smaller and in some aspects incomplete concerning sample collectionand sample control. The first study only included pellets (FIG. 6), yetnevertheless here it was found the inverse correlation for cancerpatients. In this study a group of healthy volunteers were included,which was not the case for subsequent studies. During the second studywhich did take into account supernatant-annexin A3 and pellet-annexin A3(albeit with sample numbers which were too small to come tostatistically significant solutions), there was a trend to highersupernatant to pellet ratios in cancer patients as compared to BPH andother non-cancers. This is perfectly in line with the first and thirdstudy, because obviously the low pellet annexin A3-amounts combine incancer patients with a slightly higher annexin A3-amount to biggerratios (FIG. 7). In non-cancers (like, e.g., BPH, fibrosis and others),obviously in total the considerably larger annexin A3-amounts in pelletsand supernatants combine to lower overall ratios. The robustness of theannexin A3-signal in supernatants provides an experimental and clinicaladvantageous and easy diagnostic read-out.

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What is claimed is:
 1. A method of diagnosing prostate and/or coloncancer and/or discrimination between prostate and/or colon cancerous andnon-cancerous tissue, comprising: determining abundance of extracellularhuman annexin A3 protein, in urine samples or fractions thereat whereinthe urine samples or fractions thereof are subjected to a separationprocess to yield cell pellets and supernatants prior to determining theabundance of human annexin A3 protein, whereby the diagnosis of prostateand/or colon cancer and/or discrimination between prostate and/or coloncancerous and non-cancerous tissue is made by using the supernatants todetermine the extracellular abundance of human annexin A3 protein. 2.The method according to claim 1, wherein the abundance of annexin A3protein is determined together with the abundance of at least anotherannexin protein.
 3. The method according to claim 2, wherein the atleast another annexin protein is at least one selected from the groupconsisting of annexin A1, annexin A2, annexin A4, annexin A5, annexinA6, annexin A7, annexin A8 and annexin A10 and wherein the abundance ofthe at least one annexin protein is determined together with theabundance of at least a further protein.
 4. The method according toclaim 1, wherein the abundance of at annexin A3 protein is determinedtogether with the abundance of at least one further protein selectedfrom the group consisting of serum amyloid P, isopeptidase T,muscle-type fatty acid binding protein, galectin 1, heat shock protein90, BiP, protein disulfide isomerase, epidermal-type fatty acid bindingprotein, enoyl coenzyme A hydratase and nucleophosmin.
 5. The methodaccording to claim 1, wherein the abundance of annexin A3 protein isdetermined together with the abundance of at least one protein selectedfrom the group consisting of a 14-3-3 family protein, a proteasomeprotein, activator subunit 2 protein, a cytokeratin family protein, aKNP-1 alpha protein and a KNP-1 beta protein.
 6. The method according toclaim 1, wherein the abundance of annexin A3 protein is determinedtogether with the abundance of at least one blood or serum markerselected from the group consisting of a Kallikrein protease familyprotein and prostate specific antigen (PSA).
 7. The method according toclaim 1, wherein the abundance of annexin A3 protein is determinedtogether with the abundance of at least prostate specific membraneantigene (PSMA).
 8. The method according to claim 1, wherein theabundance of annexin A3 protein is determined together with theabundance of an epithelial cell marker.
 9. The method according to claim1, wherein the at least another annexin protein is at least one selectedfrom the group consisting of annexin A4 and annexin A8.
 10. The methodaccording to claim 1, wherein the pellets are used to determine theintracellular abundance of annexin A3 protein.
 11. The method accordingto claim 1, wherein EDTA and/or EGTA is added to the urine or fractionthereof prior to determining the abundance of at least one annexinprotein.
 12. The method according to claim 1, wherein a cation chelatoradded to the urine or fraction thereof prior to determining theabundance of annexin A3 protein.
 13. The method according to claim 1,wherein the abundance of annexin A3 protein is determined byimmuno-histochemical methods.
 14. The method according to claim 1,wherein the urine samples or fractions thereof are obtained fromexprimate urine which is recovered subsequent to prostate massage. 15.The method according to claim 1, wherein the urine samples or fractionsthereof are obtained from exprimate urine which is recovered subsequentto a prostate massage performed by rectal finger insertion.
 16. Themethod according to claim 1, wherein the urine samples or fractionsthereof are purified and are free from neutrophils, monocytes orperipheral blood mononuclear cells (PMBCs).
 17. The method according toclaim 1, wherein the urine samples or magnetic fractions thereof arepurified by means of magnetic beads and are free from neutrophils,monocytes or peripheral blood mononuclear cells (PMBCs).
 18. The methodaccording to claim 1, wherein the urine samples or fractions thereof arefrom morning urine.
 19. The method according to claim 1, wherein annexinA3 protein levels are further measured in faeces or epithelial cells ofthe intestinal tract.
 20. The method according to claim 1, whereinannexin A3 protein levels are used to diagnose colon cancer.